The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 6730 -6737
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rushworth, S. A.
Right arrow Articles by O'Connell, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rushworth, S. A.
Right arrow Articles by O'Connell, M. A.

Lipopolysaccharide-Induced Expression of NAD(P)H:Quinone Oxidoreductase 1 and Heme Oxygenase-1 Protects against Excessive Inflammatory Responses in Human Monocytes

Stuart A. Rushworth*,{dagger}, David J. MacEwan* and Maria A. O'Connell1,*,{dagger}

* School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, United Kingdom; and {dagger} Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Monocytes play a central role in the immunopathological effects of sepsis. This role is mediated by production of the cytokines TNF-{alpha} and IL-1β. The transcription factor NF-E2-related factor 2 (Nrf2) regulates innate immune responses in various experimental disease models. Presently, the role of Nrf2-regulated genes in LPS-treated human monocytes is not well defined. Herein we show that Nrf2 mediates a significant regulation of LPS-induced inflammatory responses. Analysis of Nrf2-regulated gene expression in human monocytes showed that LPS induced the expression of the phase II detoxification gene NAD(P)H:quinone oxidoreductase 1 (NQO1). Furthermore, NQO1 mRNA or protein expression in response to LPS was regulated by Nrf2. Silencing Nrf2 expression in human monocytes inhibited LPS-induced NQO1 expression; however, in contrast, it significantly increased TNF and IL-1β production. Silencing expression of NQO1 alone, or in combination with heme oxygenase-1 (HO-1) silencing, markedly increased LPS-induced TNF and IL-1β expression. Additionally, overexpression of NQO1 and/or HO-1 inhibited LPS-induced TNF and IL-1β expression. These results show for the first time that LPS induces NQO1 and HO-1 expression in human monocytes via Nrf2 to modulate their inflammatory responsiveness, thus providing novel potential therapeutic strategies for the treatment of sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a major public health problem throughout the world. It affects 30–50% of intensive care unit patients (1, 2, 3), with an annual incidence of ~250 cases per 100,000 population in the Western world (4). Sepsis is characterized by a systemic inflammatory response caused by bacterial infection or trauma. The most common bacterial component implicated in initiating the septic syndrome is a cell wall molecule derived from Gram-negative bacteria, known as LPS (5). The inflammatory responses induced by LPS are predominantly controlled by monocytes and macrophages that produce the prototypic inflammatory cytokines TNF-{alpha} and IL-1β, and these in turn mediate many of the immunopathological features of sepsis (6, 7).

Host factors that regulate inflammatory responses may protect against dysfunctional inflammation during bacterial infection. The basic leucine zipper transcription factor NF-E2-related factor 2 (Nrf2),2 which, when activated, induces the expression of a host of cytoprotective and detoxification genes, is a known regulator of inflammatory responses (8, 9, 10). In endothelial cells, Nrf2 activation inhibits proinflammatory cytokine-induced adhesion molecule expression (11). In a mouse model of experimental sepsis, exposure of Nrf2-deficient mice to endotoxin leads to increased TNF and IL-6 expression when compared with wild-type animals (12). Additionally, the synthetic triterpenoid CDDO-Im (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid imidazolide) inhibits LPS-induced inflammation by inducing Nrf2-regulated genes in mice (13). Moreover, activation of Nrf2 protects inflammatory cells from oxidant injury and inhibits proinflammatory gene expression (14). Taken together, these studies suggest that Nrf2 plays an important role in modulating the magnitude of inflammatory responses. However, the mechanism by which Nrf2 mediates this protection remains to be elucidated.

Monocytes play a key role in mobilization of the immune response during sepsis (15). In response to LPS, monocytes produce both proinflammatory mediators and regulatory proteins that counteract the inflammation and oxidative stress. Indeed, we have demonstrated that LPS activation of human monocytes induces the expression of the cytoprotective protein heme oxygenase-1 (HO-1) (16). HO-1 induction was shown to be induced by Nrf2 activation. Additionally, others have shown that exposure of HO-1-deficient mice to endotoxin leads to increased splenic proinflammatory cytokine secretion, as well as higher mortality from endotoxic shock when compared with wild-type animals (17, 18). Taken together, these studies suggest that HO-1 and its transcription factor Nrf2 play an important role in counteracting deleterious increases in inflammation and oxidative injury.

Nrf2 is ubiquitously expressed in animal tissues, and increasing evidence reveals that Nrf2 mediates antioxidant response element (ARE)-dependent gene transcription (8). The genes regulated by Nrf2 include phase II enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1), GST, and glutathione reductase (GR), which detoxify endogenous and exogenous chemicals through reduction and conjugation reactions (19). It also includes the regulation of glutamyl cysteine ligase modulatory (GCLM) and catalytic (GCLC) units, the two subunits of the rate-limiting enzyme in glutathione biosynthesis. Several of these Nrf2-regulated genes have been shown to have antiinflammatory actions (8, 14, 20, 21), which suggests that Nrf2 mediates its control on inflammation by inducing the expression of any number of ARE-dependent genes.

The molecular mechanism of Nrf2 action is yet to be fully resolved. There are two reported mechanisms proposed regarding Nrf2 activation. First, Nrf2 is bound to its repressor Keap1, which inhibits its activity, and upon activation Nrf2 is released from Keap1 and translocates into the nucleus (22, 23). The second mechanism is that under normal basal conditions Keap1 regulates the ubiquitin-26S proteasome-mediated turnover of Nrf2. Upon activation, Keap1 stabilizes Nrf2, which translocates into the nucleus (24, 25). Once in the nucleus Nrf2 forms a heterodimer with Maf proteins (26). The heterodimer then binds to ARE located in the enhancer regions and mediates the transcription of the Nrf2-inducible genes (27).

In the present study, we report that exposure of human monocytes to LPS activates Nrf2 transcriptional activity and increases the expression of NQO1. We further demonstrate that transcriptional activity of Nrf2 is mediated by Nrf2 mRNA expression induced by LPS. Previously we have shown that LPS can induce the expression of HO-1 in human monocytes (16). Herein we show that silencing the expression of NQO1 and HO-1 prolongs LPS-induced TNF and IL-1β expression. These data suggest that LPS exerts protective effects through the coordinated expression of Nrf2-regulated genes in human monocytes. These findings provide an important new insight into the regulation of inflammation in human monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

The human monocytic cell line THP-1 was obtained from the European Collection of Cell Cultures. Human HO-1 Ab was purchased from StressGen Biotechnologies. All other Abs were obtained from Santa Cruz Biotechnology. LPS was procured from Calbiochem. Nrf2, NQO1, and HO-1 siRNA were purchased from Ambion. All other reagents were obtained from Sigma-Aldrich.

Cell culture

Primary human monocytes and THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine (Invitrogen), and 2-ME. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2. For monocyte isolation, heparinized blood was collected from healthy volunteers and human PBMCs were isolated by Percoll (Amersham Pharmacia Biotech) density gradient centrifugation (28). PBMCs (4 x 106/ml) were incubated in complete medium for 2 h at 37°C to allow adherence of monocytes (29). Cell type was confirmed by microscopy and flow cytometry.

Plasmids

To construct the HO-1 mammalian expression vectors, full-length human HO-1 was generated by RT-PCR and cloned into pcDNA3 to generate pcDNA3HO-1. pcDNA3NQO1 was kindly provided by X.-L. Chen (AtheroGenics) (11).

RNA extraction and real-time PCR

Total RNA was extracted from 5 x 105 treated THP-1 or primary monocytic cells using the Nucleic Acid PrepStation from Applied Biosystems, according to the manufacturer’s instructions. Reverse transcription was performed using the RNA PCR core kit (Applied Biosystems). Real-time PCR primers for GAPDH, HO-1, IL-1β, NQO1, GCLM, GSTA1, GR, Nrf2, TNF, thioredoxin reductase 1 (TrxR1), GCLC, GSTA2, and IL-10 were purchased from Invitrogen. Sequences of primers were as follows: GAPDH forward, 5'-ACCAGCCTCAAGATCATCAGCA-3', GAPDH reverse, 5'-TGCTAAGCAGTTGGTGGTGC-3'; HO-1 forward, 5'-ATGGCCTCCCTGTACCACATC-3', HO-1 reverse, 5'-TGTTGCGCTCAATCTCCTCCT-3'; IL-1β forward 5'-CTGGACCTCTGCCCTCTGG-3', IL-1β reverse 5'-TCCATGGCCACAACAACTGA-3'; NQO1 forward, 5'-CGCAGACCTTGTGATATTCCAG-3', NQO1 reverse, 5'-CGTTTCTTCCATCCTTCCAGG-3'; GCLM forward, 5'-TGCAGTTGACATGGCCTGTT-3', GCLM reverse, 5'-TCACAGAATCCAGCTGTGCAA-3'; GSTA1 forward, 5'-AGCCCAAGCTCCACTACTTCAAT-3', GSTA1 reverse, 5'-CTTCAAACTCTACTCCAGCTGCAG-3'; GR forward, 5'-TTGTCGGGCTTGGAAGTCAG-3', GR reverse, 5'-TGGTAGCCTACCGGGAACTG-3'; Nrf2 forward, 5'-AACCACCCTGAAAGCACAGC-3', Nrf2 reverse, 5'-TGAAATGCCGGAGTCAGAATC-3'; TNF forward, 5'-GCCCAGGCAGTCAGATCATC-3', TNF reverse, 5'-CGGTTCAGCCACTGGAGCT-3'; TrxR1 forward, 5'-GGGCAATTTATTGGTCCTCA-3, TrxR1 reverse, 5'-GGTCTTTCACCAGTGGCAAT-3; GCLC forward, 5'-GGATTTGGAAATGGGCAATTG-3', GCLC reverse, 5'-CTCAGATATACTGCAGGCTTGGAA-3'; GSTA2 forward, 5'-ATGTTCCAGCAAGTGCCAATG-3', GSTA2 reverse, 5'-TGAGAATGGCTCTGGTCTGCA-3'; IL-10 forward, 5'-TTACCTGGAGGAGGTGATGC-3', IL-10 reverse, 5'-GGCCTTGCTCTTGTTTTCAC-3'. Relative quantitative real-time PCR used SYBR Green technology (Sigma-Aldrich) on cDNA generated from the reverse transcription of purified RNA. After preamplification (95°C for 2 min), the PCRs were amplified for 40 cycles (95°C for 15 s and 60°C for 1 min) on a IQ5 real-time PCR detection system (Bio-Rad). Each mRNA expression was normalized against GAPDH mRNA expression using the comparative cycle threshold method.

Protein expression

TNF-{alpha} protein expression was measured using the human TNF-{alpha} ELISA set (BD Biosciences). SDS-PAGE and Western analyses were performed as described previously (29, 30). Nuclear extracts were prepared as described previously (31). Protein was transferred to nitrocellulose membrane, and Western analyses were performed with the indicated Ab according to their manufacturers’ guidelines, using ECL detection. In some instances, immunoblot band intensity was measured by densitometric analysis using Quantity One software (Bio-Rad).

DNA binding assays

NF-{kappa}B DNA binding was measured using the NF-{kappa}B p65 transcription factor ELISA kit (Panomics). Nrf2 binding was measured by EMSA. An oligonucleotide probe containing the human NQO1 ARE site (underlined) (5'-TTCCAATCCGCAGTCACAGTGACTCAGCAGA-3') was manufactured and biotinylated by Sigma-Genosys. For competition binding, the same sequence was manufactured without the biotin label (Sigma-Genosys). Nuclear extracts were prepared as previously described (16) and incubated with the biotin-labeled probes using the LightShift Chemiluminescent EMSA kit (Pierce), following the manufacturer’s instructions. For supershift analysis, nuclear extracts from treated THP-1 cells were preincubated with 1 µg of either anti-human Nrf2 or anti-human p65 supershift Abs (Santa Cruz Biotechnology) for 20 min before gel shift analysis.

Measurement of reactive oxygen species (ROS)

A dichlorofluorescein (DCF) assay was used to determine cellular ROS generation in THP-1 cells (32). Briefly, 106 cells were unstimulated or treated with 10 µg/ml LPS for various times. Following this, cells were washed in PBS and then incubated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) (Molecular Probes) for 15 min at 37°C and 5% CO2. Cells were collected and washed with RPMI 1640 containing 10 mM HEPES and measured for oxidation of H2-DCFDA using a flow cytometer (Beckman Coulter Epics XL). The fluorescence intensity measuring the oxidation of H2-DCFDA by ROS represents the relative steady-state of ROS generation in cells. Thus, H2-DCFDA oxidation was calculated by dividing the mean channel fluorescence of a treated sample by that of the untreated one and multiplying by 100 to obtain the relative change, expressed as a percentage (33).

Transfections

Primary monocytes and THP-1 cells (1 x 106/well) were transfected using Amaxa Nucleofector technology. Equivalent molar concentrations of the small interfering RNA (siRNA) were used to yield a final concentration of 30 nM. Transfected cells were incubated for 24 h. Following this, cells were stimulated as described above. THP-1 cells were transfected with various plasmids as indicated in the figure legends using 1 µg of DNA for 1 x 106 cells using Amaxa Nucleofector technology.

Statistical analyses

Student’s t test was performed to assess statistical significance from controls. Results with p < 0.05 were considered statistically significant. Results are displayed as means ± SD of at least three independent experiments. For Western blotting and EMSA experiments, data are representative of three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPS induces NQO1 expression in human monocytes

Nrf2 is a key transcription factor in the regulation of antioxidant and cytoprotective genes (8). We have recently shown that HO-1 is regulated by Nrf2 in response to LPS (16). To determine whether other Nrf2-regulated genes are induced by LPS in human monocytes, we examined the mRNA expression of GCLM, GCLC, TrxR1, HO-1, NQO1, GR, GSTA1, and GSTA2 in response to LPS stimulation at 8 and 24 h. LPS stimulated a significant increase in NQO1 mRNA expression as well as the positive control HO-1 in primary human monocytes (Fig. 1A). No significant induction of GSTA1, GSTA2, GCLC, GCLM, GR, and TrxR1 was seen (Fig. 1A). Further analysis revealed that LPS induced NQO1 expression in the human monocytic cell line THP-1 and that LPS induced NQO1 mRNA expression by 4 h, increased further by 8 h, decreased by 12 h, and further decreased by 16 and 24 h (Fig. 1B). To confirm the mRNA results, NQO1 protein expression was examined by Western blot analysis. LPS-induced NQO1 protein increased in both THP-1 cells and human monocytes (Fig. 1C). Immunoblots were reprobed with a mouse anti-human β-actin Ab to confirm equal loading between samples. These results demonstrate similar results in both primary monocytes and THP-1 cells, suggesting that the THP-1 cells are a valid model for studying LPS-induced Nrf2 activation in human monocytes.


Figure 1
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 1. LPS induces NQO1 expression in human monocytes. Monocytes and THP-1 cells were unstimulated or stimulated with LPS (10 µg/ml) for the indicated times and RNA was extracted. A, LPS-induced expression of Nrf2-regulated genes in peripheral blood-derived human monocytes measured by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. Data shown represent three experiments (mean ± SD). *, p < 0.01. B, Time course of LPS-induced expression of Nrf2-regulated genes in human monocytes and THP-1 cells measured by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. Data shown represent three experiments (mean ± SD). C, LPS-induced NQO1 protein expression in human monocytes and THP-1 cells, measured by Western blot analysis. Chemiluminescent images were quantified by densitometry. The numerical values represent fold increase in expression following stimulation with LPS compared with no LPS, and normalized to β-actin as a loading control.

 
Nrf2 mRNA and protein are up-regulated in response to LPS in THP-1 cells

How Nrf2 is activated in response to LPS is not clearly understood. As outlined above, the potential molecular mechanisms of Nrf2 activation could be either release of Nrf2 protein from its bound repressor Keap1, or the inhibition of Keap1-regulated ubiquitin-proteasome degradation of Nrf2, with either situation leading to Nrf2 translocation to the nucleus (24, 25). To address this question, we first examined the expression of Nrf2 in response to LPS in whole cell extracts to determine whether there are any changes in Nrf2 protein expression in response to LPS. Fig. 2A shows that LPS induced the expression of Nrf2 protein in human THP-1 cells, which peaks at 4 h but was still above control levels at 24 h. These results suggested that Nrf2 protein is synthesized de novo. To confirm these results we examined the accumulation of Nrf2 in response to LPS in THP-1 cells after treatment with the protein synthesis inhibitor cycloheximide (CHX). While accumulation of Nrf2 protein was induced 2 h after LPS treatment (Fig. 2B), it was completely inhibited by the treatment of CHX. Taken together, these results demonstrate that in response to LPS treatment, Nrf2 protein is synthesized de novo, rather than existing protein being liberated from Keap1. To determine whether Nrf2 was regulated at the transcriptional level, we used the RNA synthesis inhibitor actinomycin D (ActD). Fig. 2C shows that ActD completely inhibited LPS-induced Nrf2 protein expression in THP-1 cells. Keap1 expression was unchanged in response to LPS treatment in THP-1 cells (Fig. 2D), suggesting a minimal role for Keap1 in regulating LPS-induced Nrf2 activation. To confirm these results we also analyzed mRNA expression, which unexpectedly showed that LPS induced the rapid expression of Nrf2 mRNA expression in THP-1 cells (Fig. 2E). These results confirm that a third model of Nrf2 activation exists where Nrf2 transcription is increased, in this case by LPS treatment, which gives rise to transcribed Nrf2 mRNA and subsequent Nrf2 protein accumulation.


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 2. LPS induces de novo synthesis of Nrf2. THP-1 cells were stimulated with LPS (10 µg/ml) for various times and whole cell extracts were prepared and separated by SDS-PAGE for Western blot analysis of (A–C) Nrf2 or (D) Keap1 protein expression. THP-1 cells were pretreated with either (B) CHX or (C) ActD, respectively, for 1 h before LPS treatment and Western blot analysis of Nrf2 protein expression. Chemiluminescent images were quantified by densitometry. The numerical values represent fold increase in expression following stimulation with LPS compared with no LPS, and normalized to β-actin as a loading control. E, THP-1 cells were stimulated with LPS for various times and RNA extracts were prepared. Nrf2 mRNA expression was measured by real-time PCR. Data shown represent results from three experiments (mean ± SD).

 
LPS induces Nrf2 activation in THP-1 cells

Nrf2 is a key transcription factor in the regulation of cytoprotective genes including NQO1 and HO-1. Nuclear accumulation is an important mechanism for the activation of Nrf2. We have recently shown that Nrf2 accumulates in the nucleus in response to LPS up to 4 h (16). After showing that Nrf2 expression is induced by LPS, we next examined whether treatment with LPS induced Nrf2 nuclear accumulation beyond 4 h. THP-1 cells were treated with LPS for up to 12 h. Nuclear extracts were prepared and analyzed for Nrf2 expression by Western blot analysis. Fig. 3A demonstrates that Nrf2 appeared in the nucleus in response to LPS with significant accumulation by 2 h and plateauing at around 8 h of treatment. To verify these results in primary monocytes, we examined the subcellular localization of Nrf2 in response to LPS. Fig. 3B shows that Nrf2 accumulates in the nucleus and cytosol by 2 h following LPS treatment in primary monocytes. The kinetics of Nrf2 protein appearance in the nucleus coincided with binding of a complex to the NQO1 ARE (Fig. 3C). The binding of this complex was inhibited by the addition of unlabeled oligonucleotide, demonstrating specificity of the sequence (Fig. 3D). The presence of Nrf2 in the complex was confirmed by supershift analysis. Anti-p65 (Rel A NF-{kappa}B) supershift Abs were used as a negative control. The complex bound to the NQO1 ARE was abolished with anti-Nrf2 Abs but not with anti-p65 Abs (Fig. 3D).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 3. LPS induces Nrf2 activation in THP-1 cells. A, THP-1 cells were stimulated with LPS (10 µg/ml) for various times and nuclear extracts were prepared. Nuclear proteins were separated by SDS-PAGE, and Western blot analysis was conducted for Nrf2 protein expression. Chemiluminescent images were quantified by densitometry. The numerical values represent fold increase in expression following stimulation with LPS compared with no LPS, and normalized to β-actin as a loading control. B, Primary monocytes were stimulated with LPS for various times and cytosolic and nuclear extracts were prepared. Extracts were separated by SDS-PAGE, and Western blot analysis was conducted for Nrf2 protein levels. Cytosolic blots were reprobed with GAPDH and nuclear blots were reprobed with TATA-binding protein (TBP) to confirm sample loading. C, EMSAs were performed using LPS-stimulated nuclear THP-1 extracts and a biotinylated NQO1-ARE probe. D, Nuclear extracts from THP-1 cells stimulated for 2 h with LPS then preincubated with 1 µg of anti-Nrf2 or anti-p65 supershift Abs, before EMSA. An unbiotinylated NQO1-ARE oligonucleotide probe was used as a cold competitor (as indicated).

 
ROS do not play a key role in LPS-induced Nrf2 activation

Since it has been shown that Keap1 regulates the redox-sensitive activation of Nrf2 (26), and that ROS is known to be produced in response to LPS (32), the role of ROS was examined in this system. We analyzed the production of ROS in THP-1 cells using H2-DCFDA in response to LPS. H2-DCFDA oxidation occurred in a time-dependent manner (Fig. 4A), suggesting that LPS induces ROS in THP-1 cells. The kinetics of ROS activation correlated with the nuclear accumulation of Nrf2 following LPS. However, the anti-oxidant N-acetyl cysteine (NAC) had no effect on LPS-induced Nrf2 accumulation in the cytosol and only minimally inhibited nuclear accumulation (Fig. 4B), suggesting that the production of ROS by LPS is not required for Nrf2 activation.


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 4. ROS do not regulate LPS-induced Nrf2 activation. A, THP-1 cells were treated with LPS for up to 2 h, followed by washing with PBS and incubation with 10 µM of H2DCFDA for 15 min. Cells were then assessed for H2DCFDA oxidation using flow cytometry. B, THP-1 cells were pretreated with 10 mM NAC before treatment with LPS for 4 h and cytosolic and nuclear extracts were prepared. Western blot analysis was conducted using anti-Nrf2 Ab. Results are representative of similar findings from at least three separate experiments. Chemiluminescent images were quantified by densitometry. The numerical values represent fold increase in expression following stimulation with LPS or NAC compared with no LPS and no NAC, and normalized to β-actin as a loading control.

 
Silencing Nrf2 enhances LPS-induced inflammatory responses in human monocytes

The inflammatory responses induced by LPS are predominantly controlled through monocytes, which produce the prototypic inflammatory cytokines TNF and IL-1β, and these in turn mediate many of the symptoms observed during sepsis (6, 15). To understand the role of Nrf2 in modulating LPS-induced inflammation in human monocytes, Nrf2 siRNA was used to silence Nrf2 protein expression (Fig. 5A). Primary human monocytes and THP-1 cells were either untransfected or transfected with control siRNA or with two independent Nrf2-targeted siRNA for 24 h. Western blot analysis of treated samples shows that both Nrf2 siRNAs induced knockdown of the Nrf2 protein within 24 h. However, from this experiment Nrf2 siRNA 1 was deemed more effective than Nrf2 siRNA 2, and therefore this was used in subsequent experiments. To confirm that Nrf2 is responsible for the induced expression of NQO1 and HO-1 after treatment with LPS, cells were transfected with Nrf2 siRNA. Compared with control siRNA, Nrf2 siRNA 1 inhibited LPS-induced NQO1 and HO-1 mRNA expression (Fig. 5B), verifying that Nrf2 regulates LPS-induced HO-1 and NQO1 expression in human monocytes. We then examined the role of Nrf2 in regulating LPS-induced TNF and IL-1β expression during a 48-h period. Fig. 5C shows that in response to LPS, there was a significant difference in expression levels of both TNF and IL-1β mRNA levels between control siRNA and Nrf2 siRNA samples from 16 h up to and including the 32 h time point. These results suggest that Nrf2 inhibits prolonged LPS-induced proinflammatory responses and not the initial transient LPS-induced responses.


Figure 5
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 5. Silencing Nrf2 augments LPS-induced inflammatory responses in human monocytes. A, Monocytes and THP-1 cells were untransfected or transfected with 30 nM of either control siRNA, Nrf2 siRNA 1, or Nrf2 siRNA 2 for 24 h before treatment with LPS for various times. Nrf2 protein expression was measured by Western blot analysis. B and C, THP-1 cells were transfected with 30 nM of either control siRNA or Nrf2 siRNA 1 for 24 h and then stimulated with LPS for various time points. Treated cells were extracted for mRNA, reverse transcribed, and measured for (B) HO-1 or NQO1 or (C) TNF or IL-1β mRNA expression levels detected by real-time PCR. Data represent the means ± SD from three independent experiments. *, p < 0.05 between the different treatment groups.

 
Silencing HO-1 and NQO1 induction expands LPS-induced inflammatory responses

Both HO-1 and NQO1 have been shown to exert antiinflammatory properties (11, 34). To determine whether the effects seen with Nrf2 siRNA on LPS-induced TNF and IL-1β (in Fig. 5) are due to the induced expression of HO-1 and NQO1, we used siRNA to silence HO-1 and NQO1 in isolation and also in combination. Transfection of THP-1 cells with HO-1 siRNA and NQO1 siRNA alone or in combination resulted in almost complete knockdown of their respective protein expression (Fig. 6A). Furthermore, Fig. 6B shows that LPS-induced HO-1 and NQO1 mRNA expression is significantly and specifically reduced in cells transfected with either HO-1 siRNA, NQO1 siRNA, or both. Next we examined the effect of HO-1 and NQO1 siRNA on LPS-induced TNF and IL-1β mRNA expression. Fig. 6C shows that HO-1 and NQO1 siRNA alone significantly prolonged the LPS-induced TNF and IL-1β expression. Additionally, when we used HO-1 and NQO1 siRNA in combination a more significant increase in TNF and IL-1β expression resulted from the LPS treatment at any time point, when compared with control siRNA alone. Similarly, LPS-stimulated THP-1 cells transfected with siRNA targeted to Nrf2, HO-1, and NQO1 showed greater TNF protein expression when compared with control siRNA-transfected cells (Fig. 6D). In contrast, silencing HO-1, NQO1, or Nrf2 had no effect on the activation of the antiinflammatory cytokine IL-10 in THP-1 cells (data not shown). Taken together, these results demonstrate that both HO-1 and NQO1 play an important role in regulating the magnitude of LPS-induced proinflammatory responses in human monocytes.


Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 6. Silencing HO-1 and NQO1 induction in human monocytes enhances LPS-induced inflammatory responses. THP-1 cells were transfected with 30 nM of either control siRNA, HO-1 siRNA, NQO1 siRNA, Nrf2 siRNA, or HO-1 siRNA plus NQO1 siRNA in combination. A, Protein expression was measured for HO-1 and NQO1 by Western blot analysis. After transfection with siRNA, THP-1 cells were stimulated with LPS for various indicated times and RNA extracts were prepared to measure (B) HO-1 and NQO1 mRNA expression, (C) TNF and IL-1β mRNA expression, or (D) TNF protein expression. *, p < 0.05 and **, p < 0.01 significance between the different treatment groups. E, THP-1 cells were transfected with 30 nM of either control siRNA, HO-1 siRNA, NQO1 siRNA, or Nrf2 siRNA for 24 h then stimulated with LPS for 30 min and nuclear extracts were prepared. NF-{kappa}B DNA binding was assessed using the Panomics p65 NF-{kappa}B transcription factor ELISA.

 
Silencing Nrf2 and HO-1 results in enhanced LPS-induced NF-{kappa}B activation

NF-{kappa}B is known to regulate the expression of several genes that are essential for initiating and promoting inflammation, including TNF and IL-1β (15). Since silencing Nrf2, HO-1, and NQO1 in monocytes enhanced LPS-induced proinflammatory gene expression, their effects on NF-{kappa}B activation were then studied. Following LPS stimulation, NF-{kappa}B activation was examined 30 min after LPS stimulation in THP-1 cells using a p65 DNA-binding assay. Silencing HO-1 or Nrf2 resulted in significantly enhanced LPS-induced p65 DNA binding, suggesting an inhibitory role of Nrf2 and HO-1 on NF-{kappa}B activation (Fig. 6E). Interestingly, silencing NQO1 did not affect LPS-induced p65 DNA-binding, suggesting that the antiinflammatory effects of NQO1 are NF-{kappa}B-independent.

Overexpression of HO-1 and NQO1 inhibits LPS-induced activation

To confirm that HO-1 and NQO1 inhibit LPS-induced TNF and IL-1β expression, we overexpressed HO-1 and NQO1 in THP-1 cells and then examined these cells for IL-1β and TNF expression in response to LPS. Fig. 7A demonstrates that overexpression of pHO-1 or pNQO1 in THP-1 cells resulted in their respective protein expression. Fig. 7B shows the expression of TNF and IL-1β in response to LPS in cells overexpressing either HO-1 alone, NQO1 alone, or HO-1 and NQO1 in combination. Transfection with pcDNA3.1 vector alone was used as a control. Results demonstrated that LPS-induced TNF and IL-1β were significantly inhibited by either HO-1 alone or NQO1 alone. Moreover, when used in combination, HO-1 and NQO1 overexpression had an additive effect on the inhibition of LPS-induced TNF and IL-1β expression. Taken together, these results demonstrate that HO-1 and NQO1 alone and in combination modulate LPS-induced proinflammatory responses.


Figure 7
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 7. Overexpression of HO-1 and NQO1 inhibits LPS-induced cytokine expression. THP-1 cells were transiently transfected with 1 µg of either control vector (pcDNA3), HO-1 (pcDNA3-HO-1), NQO1 (pcDNA3-NQO1), or both HO-1 and NQO1 for 24 h. A, Protein extracts were prepared and analyzed for HO-1 and NQO1 overexpression. B, After transfection, cells were untreated or treated with LPS for various indicated times. RNA was extracted, and after reverse transcription, real-time PCR was used to analyze TNF and IL-1β mRNA expression levels. Data shown represents means ± SD (n = 4).

 
Sulforaphane preinduction of HO-1 and NQO1 gene expression protects against LPS-induced inflammatory responses

The dietary antioxidant sulforaphane has previously been shown to activate the Nrf2 pathway (10). To determine whether sulforaphane can induce HO-1 and NQO1 expression in human monocytes, THP-1 cells were treated with sulforaphane for up to 8 h. Fig. 8, A and B, demonstrates that sulforaphane induced NQO1 and HO-1 mRNA and protein expression in a time-dependent manner. To establish if sulforaphane protects against LPS-induced inflammatory responses, THP-1 cells were pretreated with sulforaphane for 4 h to induce HO-1 and NQO1 expression before activation with LPS for 4 h. Fig. 8C demonstrates that sulforaphane inhibited LPS-induced TNF and IL-1β mRNA expression. To determine whether NF-{kappa}B was involved in this pathway, the effect of sulforaphane on LPS-induced NF-{kappa}B DNA binding was examined. Sulforaphane suppressed LPS-induced p65 DNA-binding (Fig. 8D). Taken together, these results suggest that preactivating the Nrf2 pathway with sulforaphane inhibits LPS-induced proinflammatory responses in monocytic cells.


Figure 8
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 8. Sulforaphane inhibits LPS-induced proinflammatory responses. A, THP-1 cells were stimulated with 10 µM sulforaphane for various time points. RNA was extracted and, after reverse transcription, real-time PCR was used to analyze HO-1 and NQO1 mRNA expression levels. B, Sulforaphane-induced HO-1 and NQO1 protein expression in THP-1 cells, measured by Western blot analysis. C, THP-1 cells were preincubated with 10 µM sulforaphane for 4 h before activation with LPS for 4 h. RNA was extracted and, after reverse transcription, real-time PCR was used to analyze IL-1β and TNF-{alpha} mRNA expression levels. D, THP-1 cells were preincubated with 10 µM sulforaphane for 4 h before activation with different concentrations of LPS for 4 h and nuclear extracts were prepared. NF-{kappa}B DNA binding was assessed using the Panomics p65 NF-{kappa}B transcription factor ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Understanding host responses that modulate inflammation during sepsis is key to developing new therapeutic strategies to reduce the increasing incidence of this complex disease in developed countries. The present study demonstrates that Nrf2 induces the phase II enzyme NQO1 that in combination with HO-1 modulates LPS-induced inflammatory responses in human monocytes. Most research to date regarding the importance of Nrf2 in regulating inflammatory responses has focused on rodent models and the induction of the cytoprotective gene HO-1 (12, 17, 18). This is the first time inducible NQO1 expression has been shown to modulate IL-1β and TNF up-regulation.

The regulation of the phase II enzyme NQO1 in monocytes in response to LPS and other proinflammatory stimuli has not been studied. In this report gene expression profiling demonstrated that LPS unexpectedly induced the expression of NQO1 in primary monocytes and THP-1 cells. The expressions of other Nrf2-regulated genes including GCLM, GCLC, GSTA1, GSTA2, TrxR1, and GR were examined in response to LPS. No significant induction was observed in any of the genes associated with glutathione synthesis. This is an important finding since the glutathione system has been linked with regulating airway inflammation in mice (35).

Since NQO1 has been shown to be regulated by Nrf2, AP-1, and NF-{kappa}B (36, 37, 38), and because all of these transcription factors are known to be activated in response to LPS in human monocytes (16, 39), we examined the mechanism by which LPS induced NQO1 expression. We found that LPS-induced NQO1 expression was regulated by Nrf2. Monocytes therefore induce the expression of both HO-1 and NQO1 in response to LPS through the activation of Nrf2. This suggests that Nrf2 mediates its antiinflammatory effects by inducing the expression of NQO1, in addition to HO-1, whose role it is to protect monocytes against excessive activation.

NQO1 has been shown to have both antiinflammatory and proinflammatory properties in different cell systems (11, 40, 41). Jaiswal and coworkers have recently documented that NQO1 plays an important role in TNF-induced NF-{kappa}B activation (40), in which NQO1 is thought to modulate NF-{kappa}B through its control of redox status. In contrast, other groups have shown that NQO1 has antiinflammatory properties. Chen et al. described how NQO1 can inhibit TNF-induced VCAM-1 expression in human endothelial cells (11). Interestingly, it was reported that this regulation of TNF-induced VCAM-1 expression was not through NF-{kappa}B inhibition (11, 41). Thimmulappa et al. reported that CDDO-Im (a synthetic triterpenoid) induces NQO1 expression in neutrophils, which subsequently inhibited LPS-induced IL-6 and TNF induction (13). The results of the present study show that overexpression of NQO1 alone as well as in combination with HO-1 can inhibit LPS-induced TNF and IL-1β expression in human THP-1 cells and that this inhibition is NF-{kappa}B-independent. Taken together, these studies suggest a multifunctional role for NQO1 in modulating inflammation.

We have previously shown that LPS induces HO-1 in human monocytes through the activation of Nrf2 (16). In this study we examined the function of this induction by targeting HO-1 for gene silencing as well as overexpression studies. The results demonstrated that by silencing HO-1 expression an excessive inflammatory response in human monocytes is induced in response to LPS. Moreover, when we overexpressed HO-1 we observed that the inflammatory response induced by LPS was inhibited. Furthermore, when combining HO-1 with NQO1 silencing (Fig. 5), we observed an increase in LPS-induced TNF and IL-1β expression above silencing HO-1 or NQO1 alone. Additionally, when we overexpressed a combination of both NQO1 and HO-1 we observed a significant decrease in LPS-induced TNF and IL-1β expression. When we silenced Nrf2 we detected a similar response to that seen with NQO1 and HO-1 silenced together. Collectively, these results suggest that Nrf2 mediates its antiinflammatory effects by inducing the expression of HO-1 and NQO1, which in turn regulate the strength of the inflammatory response in human monocytes.

Studies have shown that HO-1 plays an important role in modulating inflammatory responses (42, 43, 44). Many antiinflammatory mediators, including IL-10 and 15-deoxy-prostaglandin J2, have been shown to up-regulate HO-1, which subsequently inhibits inflammation (44, 45). The antiinflammatory effects of HO-1 are thought to be mediated by its metabolites carbon monoxide and bilirubin, which have been reported to inhibit NF-{kappa}B activation in various cells and tissues (44, 45). Here, we found that overexpression of HO-1 and NQO1 can inhibit LPS-induced TNF and IL-1β expression. However, HO-1 inhibits LPS-induced TNF and IL-1β expression through suppression of NF-{kappa}B, while NQO1 inhibits this response through an NF-{kappa}B-independent pathway. Further work is required to establish this important mechanistic difference, as we may be able to use this diversity between HO-1 and NQO1 in inhibiting inflammatory responses to our advantage in a therapeutic setting.

The mechanism by which Nrf2 becomes activated in response to its many inducers is a matter of much discussion (22, 23, 24, 25, 26, 27). This report adds a new insight to that discussion since we have observed that upon activation with LPS, Nrf2 mRNA is synthesized de novo and is not dependent on production of ROS. Nrf2 protein is subsequently transcribed and functions to induce NQO1 and HO-1 expression. This suggests that Keap1 is either redundant in this system or that it is there to stabilize the transcribed Nrf2 protein, before it moves into the nucleus. This raises a number of different questions. First, what transcription machinery controls Nrf2 mRNA-induced expression? Second, can we target this transcriptional machinery for therapeutic intervention? These are questions that need to be addressed, as this information may help provide novel therapies for the treatment of sepsis as well as other diseases associated with Nrf2.

The results of the current study show that in human monocytes up-regulation of NQO1 and HO-1 by LPS is dependent on Nrf2. Furthermore, we report that LPS-induced Nrf2 activation protects monocytes from excessive inflammatory responses. Results were similar in primary monocytes and THP-1 cells, indicating that THP-1 cells are a valid model for studying Nrf2 activation as well as NQO1 and HO-1 gene expression in human monocytes. Nrf2 deficiency results in increased mortality from LPS in experimental models of sepsis. Additionally, we have shown here that the Nrf2-regulated genes NQO1 and HO-1 protect LPS-activated monocytes from excessive inflammatory responses. Moreover, we also describe a novel mechanism for the activation of Nrf2 by LPS (Fig. 9). Taken together, further understanding of the transcriptional machinery that regulates Nrf2 and its gene products may provide a novel therapeutic target for the treatment of sepsis.


Figure 9
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 9. Feedback control of LPS-induced inflammatory responses in human monocytes by inducible Nrf2.

 

   Acknowledgments
 
The authors thank Dr. D. Sexton (University of East Anglia) for the TNF-{alpha} ELISA.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Maria O'Connell, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. E-mail address: m.oconnell{at}uea.ac.uk Back

2 Abbreviations used in this paper used: Nrf2, NF-E2-related factor 2; ActD, actinomycin D; ARE, antioxidant response element; CHX, cycloheximide; GCLC, glutamyl cysteine ligase catalytic unit; GCLM, glutamyl cysteine ligase modulatory unit; GR, glutathione reductase; H2DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; HO-1, heme oxygenase-1; NAC, N-acetyl cysteine; NQO1, NAD(P)H:quinone oxidoreductase 1; ROS, reactive oxygen species; siRNA, small interfering RNA; TrxR1, thioredoxin reductase-1. Back

Received for publication May 6, 2008. Accepted for publication September 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sands, K. E., D. W. Bates, P. N. Lanken, P. S. Graman, P. L. Hibberd, K. L. Kahn, J. Parsonnet, R. Panzer, E. J. Orav, D. R. Snydman, et al 1997. Epidemiology of sepsis syndrome in 8 academic medical centers. J. Am. Med. Assoc. 278: 234-240. [Abstract/Free Full Text]
  2. Alberti, C., C. Brun-Buisson, H. Burchardi, C. Martin, S. Goodman, A. Artigas, A. Sicignano, M. Palazzo, R. Moreno, R. Boulme, et al 2002. Epidemiology of sepsis and infection in ICU patients from an international multicentre cohort study. Intensive Care Med. 28: 108-121. [Medline]
  3. Vincent, J. L., D. J. Bihari, P. M. Suter, H. A. Bruining, J. White, M. H. Nicolaschanoin, M. Wolff, R. C. Spencer, M. Hemmer. 1995. The prevalence of nosocomial infection in intensive care units in Europe: results of the European Prevalence of Infection in Intensive Care (EPIC) Study: EPIC International Advisory Committee. J. Am. Med. Assoc. 274: 639-644. [Abstract/Free Full Text]
  4. Martin, G. S., D. M. Mannino, S. Eaton, M. Moss. 2003. The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 348: 1546-1554. [Abstract/Free Full Text]
  5. Rietschel, E. T., H. Brade. 1992. Bacterial endotoxins. Sci. Am. 267: 54-61. [Medline]
  6. Cohen, J.. 2002. The immunopathogenesis of sepsis. Nature 420: 885-891. [Medline]
  7. Sweet, M. J., D. A. Hume. 1996. Endotoxin signal transduction in macrophages. J. Leukocyte Biol. 59: 280-286. [Abstract]
  8. Ishii, T., K. Itoh, S. Takahashi, H. Sato, T. Yanagawa, Y. Katoh, S. Bannai, M. Yamamoto. 2000. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem. 275: 16023-16029. [Abstract/Free Full Text]
  9. Lee, J. M., M. J. Calkins, K. M. Chan, Y. W. Kan, J. A. Johnson. 2003. Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J. Biol. Chem. 278: 12029-12038. [Abstract/Free Full Text]
  10. Jeong, W. S., M. Jun, A. N. Kong. 2006. Nrf2: a potential molecular target for cancer chemoprevention by natural compounds. Antioxid. Redox Signal. 8: 99-106. [Medline]
  11. Chen, X. L., S. E. Varner, A. S. Rao, J. Y. Grey, S. Thomas, C. K. Cook, M. A. Wasserman, R. M. Medford, A. K. Jaiswal, C. Kunsch. 2003. Laminar flow induction of antioxidant response element-mediated genes in endothelial cells: a novel anti-inflammatory mechanism. J. Biol. Chem. 278: 703-711. [Abstract/Free Full Text]
  12. Thimmulappa, R. K., H. Lee, T. Rangasamy, S. P. Reddy, M. Yamamoto, T. W. Kensler, S. Biswal. 2006. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J. Clin. Invest. 116: 984-995. [Medline]
  13. Thimmulappa, R. K., R. J. Fuchs, D. Malhotra, C. Scollick, K. Traore, J. H. Bream, M. A. Trush, K. T. Liby, M. B. Sporn, T. W. Kensler, S. Biswali. 2007. Preclinical evaluation of targeting the Nrf2 pathway by triterpenoids (CDDO-Im and CDDO-Me) for protection from LPS-induced inflammatory response and reactive oxygen species in human peripheral blood mononuclear cells and neutrophils. Antioxid. Redox Signal. 9: 1963-1970. [Medline]
  14. Guo, R. F., P. A. Ward. 2007. Role of oxidants in lung injury during sepsis. Antioxid. Redox Signal. 9: 1991-2002. [Medline]
  15. Guha, M., N. Mackman. 2001. LPS induction of gene expression in human monocytes. Cell. Signal. 13: 85-94. [Medline]
  16. Rushworth, S. A., X. L. Chen, N. Mackman, R. M. Ogborne, M. A. O'Connell. 2005. Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic cells is mediated via Nrf2 and protein kinase C. J. Immunol. 175: 4408-4415. [Abstract/Free Full Text]
  17. Chen, Y. H., S. F. Yet, M. A. Perrella. 2003. Role of heme oxygenase-1 in the regulation of blood pressure and cardiac function. Exp. Biol. Med. 228: 447-453. [Abstract/Free Full Text]
  18. Poss, K. D., S. Tonegawa. 1997. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. USA 94: 10925-10930. [Abstract/Free Full Text]
  19. Thimmulappa, R. K., K. H. Mai, S. Srisuma, T. W. Kensler, M. Yamamato, S. Biswal. 2002. Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res. 62: 5196-5203. [Abstract/Free Full Text]
  20. Otterbein, L. E., A. M. K. Choi. 2000. Heme oxygenase: colors of defense against cellular stress. Am. J. Physiol. 279: L1029-L1037.
  21. Ishii, Y., K. Itoh, Y. Morishima, T. Kimura, T. Kiwamoto, T. Ilzuka, A. E. Hegab, T. Hosoya, A. Nomura, T. Sakamoto, et al 2005. Transcription factor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysema. J. Immunol. 175: 6968-6975. [Abstract/Free Full Text]
  22. Bloom, D. A., A. K Jaiswal. 2003. Phosphorylation of Nrf2 at Ser40 by protein kinase C in response to antioxidants leads to the release of Nrf2 from INrf2, but is not required for Nrf2 stabilization/accumulation in the nucleus and transcriptional activation of antioxidant response element-mediated NAD(P)H:quinone oxidoreductase-1 gene expression. J. Biol. Chem. 278: 44675-44682. [Abstract/Free Full Text]
  23. Huang, H. C., T. Nguyen, C. B. Pickett. 2002. Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J. Biol. Chem. 277: 42769-42774. [Abstract/Free Full Text]
  24. McMahon, M., K. Itoh, M. Yamamoto, J. D. Hayes. 2003. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J. Biol. Chem. 278: 21592-21600. [Abstract/Free Full Text]
  25. Zhang, D. D., S. C. Lo, J. V. Cross, D. J. Templeton, M Hannink. 2004. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol. Cell. Biol. 24: 10941-10953. [Abstract/Free Full Text]
  26. Kimura, M., T. Yamamoto, J. Zhang, K. Itoh, M. Kyo, T. Kamiya, H. Aburatani, F. Katsuoka, H. Kurokawa, T. Tanaka, et al 2007. Molecular basis distinguishing the DNA binding profile of Nrf2-Maf heterodimer from that of Maf homodimer. J. Biol. Chem. 282: 33681-33690. [Abstract/Free Full Text]
  27. Itoh, K., T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, et al 1997. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236: 313-322. [Medline]
  28. Boyum, A.. 1968. Isolation of mononuclear cells and granulocytes from human blood: isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 21: 77-89.
  29. O'Connell, M. A., B. L. Bennett, F. Mercurio, A. M. Manning, N. Mackman. 1998. Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells. J. Biol. Chem. 273: 30410-30414. [Abstract/Free Full Text]
  30. Zhou, Z., M. C. Connell, D. Mac Ewan. 2007. TNFR1-induced NF-{kappa}B, but not ERK, p38 MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells. Cell. Signal. 19: 1238-1248. [Medline]
  31. Cordle, S. R., R. Donald, M. A. Read, J. Hawiger. 1993. Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-{kappa}B proteins in human monocytic THP-1 cells. J. Biol. Chem. 268: 11803-11810. [Abstract/Free Full Text]
  32. Chi, L., Y. Ke, C. Luo, D. Gozal, R. Liu. 2007. Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo. Neuroscience 144: 991-1003. [Medline]
  33. Ryan, K. A., M. F. Smith, Jr, M. K. Sanders, P. B. Ernst. 2004. Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-{kappa}B and interleukin-8 expression. Infect. Immun. 72: 2123-2130. [Abstract/Free Full Text]
  34. Rushworth, S. A., M. A. O'Connell. 2004. Haem oxygenase-1 in inflammation. Biochem. Soc. Trans. 32: 1093-1094. [Medline]
  35. Koike, Y., T. Hisada, M. Utsugi, T. Ishizuka, Y. Shimizu, A. Ono, Y. Murata, J. Hamuro, M. Mori, K. Dobashi. 2007. Glutathione redox regulates airway hyperresponsiveness and airway inflammation in mice. Am. J. Respir. Cell Mol. Biol. 37: 322-329. [Abstract/Free Full Text]
  36. He, X. Q., M. G. Chen, G. X. Lin, Q. Ma. 2006. Arsenic induces NAD(P)H-quinone oxidoreductase I by disrupting the Nrf2 x Keap1 x Cul3 complex and recruiting Nrf2 x Maf to the antioxidant response element enhancer. J. Biol. Chem. 281: 23620-23631. [Abstract/Free Full Text]
  37. Yao, K. S., P. J. O'Dwyer. 1995. Involvement of NF-{kappa}B in the induction of NAD(P)H:quinone oxidoreductase (DT-diaphorase) by hypoxia, oltipraz and mitomycin C. Biochem. Pharmacol. 49: 275-282. [Medline]
  38. Yao, K. S., P. J. O'Dwyer. 2003. Role of the AP-1 element and redox factor-1 (Ref-1) in mediating transcriptional induction of DT-diaphorase gene expression by oltipraz: a target for chemoprevention. Biochem. Pharmacol. 66: 15-23. [Medline]
  39. Yao, J., N. Mackman, T. S. Edgington, S. T. Fan. 1997. Lipopolysaccharide induction of the tumor necrosis factor-{alpha} promoter in human monocytic cells: regulation by Egr-1, c-Jun, and NF-{kappa}B transcription factors. J. Biol. Chem. 272: 17795-17801. [Abstract/Free Full Text]
  40. Iskander, K., J. Li, S. H. Han, B. Zheng, A. K. Jaiswal. 2006. NQO1 and NQO2 regulation of humoral immunity and autoimmunity. J. Biol. Chem. 281: 30917-30924. [Abstract/Free Full Text]
  41. Chen, X. L., G. Dodd, S. Thomas, X. L. Zhang, M. A. Wasserman, B. H. Rovin, C. Kunsch. 2006. Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression. Am. J. Physiol. 290: H1862-H1870.
  42. Jeon, W.-K., and B.-C. Kim. 2007. Heme oxygenase-1 mediates the anti-inflammatory effect of propyl gallate in LPS-stimulated macrophages. Biochem. Biophys. Res. Commun. July 25. epub ahead of print.
  43. Lee, T. S., L. Y. Chau. 2002. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat. Med. 8: 240-246. [Medline]
  44. Lee, T. S., H. L. Tsai, L. Y. Chau. 2003. Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-{Delta}12,14-prostaglandin J2. J. Biol. Chem. 278: 19325-19330. [Abstract/Free Full Text]
  45. True, A. L., M. Olive, M. Boehm, H. San, R. J. Westrick, N. Raghavachari, X. Xu, E. G. Lynn, M. N. Sack, P. J. Munson, et al 2007. Heme oxygenase-1 deficiency accelerates formation of arterial thrombosis through oxidative damage to the endothelium, which is rescued by inhaled carbon monoxide. Circ. Res. 101: 893-901. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rushworth, S. A.
Right arrow Articles by O'Connell, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rushworth, S. A.
Right arrow Articles by O'Connell, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS